Abstract
Cell signaling mediated by the G protein–coupled parathyroid hormone receptor type 1 (PTHR) is fundamental to bone and kidney physiology. It has been unclear how the two ligand systems—PTH, endocrine and homeostatic, and PTH-related peptide (PTHrP), paracrine—can effectively operate with only one receptor and trigger different durations of the cAMP responses. Here we analyze the ligand response by measuring the kinetics of activation and deactivation for each individual reaction step along the PTHR signaling cascade. We found that during the time frame of G protein coupling and cAMP production, PTHrP1−36 action was restricted to the cell surface, whereas PTH1−34 had moved to internalized compartments where it remained associated with the PTHR and Gαs, potentially as a persistent and active ternary complex. Such marked differences suggest a mechanism by which PTH and PTHrP induce differential responses, and these results indicate that the central tenet that cAMP production originates exclusively at the cell membrane must be revised.
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Acknowledgements
J.-P.V. thanks M.J. Lohse (University of Wuerzburg) and N.O. Nikolaev (University of Wuerzburg) for generously sharing their plasmid encoding Epac-CFP/YFP, C. Berlot (Weis Center for Research) for the plasmid encoding Gαs-CFP, M. Bünemann (University of Wuerzburg) for the Gγ2-CFP construct and A. Bisello (University of Pittsburgh) for Dyn K44A. The authors thank D. Altschuler for the use of his FRET imaging microscope. This work was supported by start-up funds from the Department of Medicine, Massachusetts General Hospital, and the Department of Pharmacology and Chemical Biology, University of Pittsburgh (to J.-P.V.). M.C. is at the Department of Pharmacology, School of Pharmacy, University of Santiago de Compostella (Spain) and received a fellowship from the Xunta de Galicia (Spain) for a research sabbatical at the J.-P.V. laboratory at the Endocrine Unit of the Massachusetts General Hospital. R.B. was supported by US National Institutes of Health grant DK38451 and received an investigator award from the National Kidney Foundation. J.-P.V. thanks P. Friedman and G. Romero for critically commenting on the manuscript.
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S.F. and T.N.F. performed most of the experiments with the support of M.C., B.W. and R.B.; J.-P.V. designed and supervised the experiments and wrote the manuscript with the support of T.J.G., J.T.P., S.F. and T.N.F.
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Supplementary Figures 1–10 and Supplementary Methods (PDF 16769 kb)
Supplementary Video 1
A 3D view of TMR-labeled ligands and GFPN-PTHR in live HEK-293 cells by spinning disc confocal microscopy 20 min after ligand wash out. PTH(1?34)TMR (red ) and GFPN-PTHR (green) co-localized within endocytic compartments. (MOV 330 kb)
Supplementary Video 2
A 3D view of TMR-labeled ligands and GFPN-PTHR in live HEK-293 cells by spinning disc confocal microscopy 20 min after ligand wash out. In contrast, PTHrP(1–36)TMR alone is detected as small puntae at internalized sites. (MOV 284 kb)
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Ferrandon, S., Feinstein, T., Castro, M. et al. Sustained cyclic AMP production by parathyroid hormone receptor endocytosis. Nat Chem Biol 5, 734–742 (2009). https://doi.org/10.1038/nchembio.206
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DOI: https://doi.org/10.1038/nchembio.206
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